Antisense modulation of purinoreceptor p2x3

ABSTRACT

The invention relates to antisense oligonucleotides, compositions and methods useful for modulating the expression of P2X 3 . The compositions comprise antisense oligonucleotides, particularly antisense oligonucleotides targeted to nucleic acids encoding P2X 3 .

RELATED APPLICATIONS

This patent application claims priority to U.S. Application No.60/337,338, filed Nov. 9, 2001.

TECHNICAL FIELD

This invention relates to antisense oligonucleotides targeted tospecific nucleotide sequences. In particular, the invention pertains toantisense oligonucleotides targeted to the nucleic acid encoding theP2X₃ purinoreceptor, and to their use for reducing cellular levels ofP2X₃.

BACKGROUND

The P2X purinoreceptors are a family of ion channels that are activatedby extracellular adenosine triphosphate (ATP). Purinoreceptors have beenimplicated in a variety of biological functions, especially thoserelated to pain sensitivity. The P2X₃ receptor subunit is a member ofthis family that was originally cloned from rat dorsal root ganglia(Chen et al. (1995) Nature 377:428-431). The nucleotide and amino acidsequences of both rat and human P2X₃ are known (Lewis et al. (1995)Nature 377:432-435; and Garcia-Guzman et al., (1997) Brain Res. Mol.Brain Res. 47:59-66). P2X₃ is involved in afferent pathways controllingurinary bladder volume reflexes. Therefore, inhibiting P2X₃ may havetherapeutic potential in the treatment of disorders of urine storage andvoiding such as overactive bladder (Cockayne et al., (2000) Nature,407:1011-5). P2X₃ also is selectively expressed on nociceptive, smalldiameter sensory neurons (i.e., neurons that are stimulated by pain orinjury), consistent with a role in pain sensitivity. A method forreducing the level or activity of P2X₃ therefore would be useful formodulating pain sensation in a subject suffering from chronic pain.

SUMMARY

Antisense oligonucleotides can be targeted to specific nucleic acidmolecules in order to reduce the expression of the target nucleic acidmolecules. For example, antisense oligonucleotides directed at the P2X₃mRNA could be used therapeutically to reduce the level of P2X₃ receptorsin a patient suffering from chronic pain. An inherent challenge ofgenerating antisense oligonucleotides, however, is identifying nucleicacid sequences that are useful targets for antisense molecules.Antisense oligonucleotides are often targeted to sequences within atarget mRNA based on, for example, the function of the sequences (e.g.,the translation start site, coding sequences, etc.). Such approachesoften fail because in its native state, mRNA is generally not in alinear conformation. Typically, mRNAs are folded into complex secondaryand tertiary structures, rendering sequences on the interior of suchfolded molecules inaccessible to antisense oligonucleotides. Onlyantisense molecules directed to accessible portions of an mRNA caneffectively contact the mRNA and potentially bring about a desiredresult. P2X₃ antisense molecules that are useful to reduce levels ofP2X₃ and alleviate pain therefore must be directed at accessible mRNAsequences. The invention described herein provides P2X₃ antisenseoligonucleotides directed to accessible portions of a P2X₃ mRNA. Theseantisense oligonucleotides are therapeutically useful for reducing P2X₃levels.

The invention features isolated antisense oligonucleotides consistingessentially of 10 to 50 nucleotides and compositions containing suchantisense oligonucleotides. The oligonucleotide can specificallyhybridize within an accessible region of the rat P2X₃ mRNA in its nativestate, wherein the accessible region is defined by nucleotides 68through 88, 209 through 230, 235 through 247, 285 through 296, 346through 355, 383 through 406, 490 through 512, 530 through 543, 553through 565, 649 through 658, 665 through 679, 727 through 739, 756through 779, 817 through 856, 874 through 912, 959 through 991, 1028through 1050, 1087 through 1116, 1145 through 1177, 1237 through 1256,1266 through 1281, 1297 through 1307, 1314 through 1334, 1339 through1359, 1434 through 1463, 1523 through 1535, 1630 through 1646, 1677through 1688, or 1729 through 1741. The antisense oligonucleotide of theinvention also can inhibit the production of P2X₃.

The isolated antisense oligonucleotide can specifically hybridize withinan accessible region defined by nucleotides 383 through 406, 756 through779, 490 through 512, or 727 through 739 of SEQ ID NO:1. The isolatedantisense oligonucleotide can specifically hybridize within anaccessible region defined by nucleotides 384 through 397, 766 through775, 495 through 510, or 732 through 736 of SEQ ID NO:1. The isolatedantisense oligonucleotide can specifically hybridize within anaccessible region defined by nucleotides 1434 through 1463, 1237 through1256, 959 through 991, or 1028 through 1050 of SEQ ID NO:1. The isolatedantisense oligonucleotide can specifically hybridize within anaccessible region defined by nucleotides 817 through 856, 553 through565, 285 through 296, 209 through 230, or 1145 through 1177 of SEQ IDNO:1. The isolated antisense oligonucleotide can specifically hybridizewithin an accessible region defined by nucleotides 383 through 404, 721through 744, 747 through 770, or 1314 through 1344 of SEQ ID NO:1.

In some embodiments, compositions include a plurality of isolatedantisense oligonucleotides, wherein each antisense oligonucleotidespecifically hybridizes within a different accessible region.

The invention also features an isolated antisense oligonucleotideconsisting essentially of 10 to 50 nucleotides, wherein theoligonucleotide specifically hybridizes within an accessible region,wherein the region is defined by nucleotides 7 through 29, 95 through105, 207 through 217, 221 through 240, 248 through 258, 278 through 293,338 through 365, 471 through 482, 486 through 502, 544 through 562, 747through 761, 784 through 796, 815 through 850, 865 through 879, 883through 905, 922 through 932, 953 through 968, 985 through 1000, 1033through 1044, 1156 through 1170, 1239 through 1261, 1297 through 1314,or 1411 through 1439 of SEQ ID NO:2, and wherein the isolated antisenseoligonucleotide inhibits the production of P2X₃. The isolated antisenseoligonucleotide can specifically hybridize within an accessible regiondefined by nucleotides 953 through 968, 1297 through 1314, or 815through 850 of SEQ ID NO:2; an accessible region defined by nucleotides957 through 967, 1297 through 1301, or 817 through 823 of SEQ ID NO:2;an accessible region defined by nucleotides 747 through 761, 985 through1000, or 486 through 502 of SEQ ID NO:2; an accessible region defined bynucleotides 338 through 365, 278 through 293, 544 through 562, or 221through 240 of SEQ ID NO:2; or within an accessible region defined bynucleotides 484 through 501 or 742 through 762 of SEQ ID NO:2.

The invention also features compositions containing such isolatedantisense oligonucleotides. The composition can include a plurality ofisolated antisense oligonucleotides, wherein each antisenseoligonucleotide specifically hybridizes with a different accessibleregion.

In another aspect, the invention features an isolated oligonucleotideconsisting essentially of the sequence of SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

In yet another aspect, the invention features a method of decreasingproduction of P2X₃ in cells or tissues. The method includes contactingthe cells or tissues with an antisense oligonucleotide that specificallyhybridizes within an accessible region of P2X₃ provided that theaccessible region is not a region defined by nucleotides 1279 through1296 or 1315 through 1334 of SEQ ID NO:2. The contacting step can resultin an inhibition of pain sensory neurons, or can result in increasedbladder capacity.

The invention also features an nucleic acid construct that includes aregulatory element operably linked to a nucleic acid encoding atranscript, wherein the transcript specifically hybridizes within one ormore accessible regions of P2X₃ mRNA in its native form, and host cellscontaining such nucleic acid constructs.

In yet another aspect, the invention features an isolated antisenseoligonucleotide that specifically hybridizes within an accessible regionof P2X₃ mRNA in its native form, provided that the accessible region isnot a region defined by nucleotides 1279 through 1296 or 1315 through1334 of SEQ ID NO:2, and wherein the antisense oligonucleotide inhibitsproduction of P2X₃.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is the nucleotide sequence of rat P2X₃ (SEQ ID NO: 1).

FIG. 2 is the nucleotide sequence of human P2X₃ (SEQ ID NO:2).

FIG. 3A and FIG. 3B are line graphs showing the result of nociceptivetesting in rats, after catheterization but before induction of chronicneuropathic pain, after induction of chronic neuropathic pain but beforeantisense treatment, and after antisense treatment. FIG. 3A depicts theresults in rats subjected to a mechanical stimulus, and FIG. 3B depictsthe results in rats subjected to a thermal stimulus.

FIG. 4A and FIG. 4B are line graphs showing the result of nociceptivetesting in rats, after catheterization but before induction of chronicneuropathic pain, after induction of chronic neuropathic pain but beforeantisense treatment, and after antisense treatment. FIG. 4A depicts theresults in rats subjected to a mechanical stimulus, and FIG. 4B depictsthe results in rats subjected to a thermal stimulus.

FIG. 5A and FIG. 5B are line graphs showing the results of nociceptivetesting in rats, after catheterization but before induction of chronicinflammatory pain, after induction of chronic inflammatory pain butbefore antisense treatment, and after antisense treatment. FIG. 5Adepicts the results in rats subjected to a mechanical stimulus, and FIG.5B depicts the results in rats subjected to a thermal stimulus.

FIG. 6A, FIG. 6A′, FIG. 6B, and FIG. 6B′ are photographs showing theimmunolocalization of P2X₃ in human spinal cord (FIG. 6A and FIG. 6A′)and human dorsal root ganglia (FIG. 6B and FIG. 6B′). For controlexperiments (shown in FIG. 6A′ and FIG. 6B′), P2X₃ antibodies werepre-incubated with the peptide antigen.

DETAILED DESCRIPTION

The present invention employs antisense compounds, particularlyoligonucleotides, to modulate the function of target nucleic acidmolecules. As used herein, the term “target nucleic acid” refers to bothRNA and DNA, including cDNA, genomic DNA, and synthetic (e.g.,chemically synthesized) DNA. The target nucleic acid can bedouble-stranded or single-stranded (i.e., a sense or an antisense singlestrand). In some embodiments, the target nucleic acid encodes a P2X₃polypeptide. Thus, a “target nucleic acid” encompasses DNA encodingP2X₃, RNA (including pre-mRNA and mRNA) transcribed from such DNA, andalso cDNA derived from such RNA. FIGS. 1 and 2 provide nucleic acidsequences that encode rat and human P2X₃ polypeptides, respectively (SEQID NO:1 and SEQ ID NO:2, respectively). An “antisense” compound is acompound containing nucleic acids or nucleic acid analogs that canspecifically hybridize to a target nucleic acid, and the modulation ofexpression of a target nucleic acid by an antisense oligonucleotide isgenerally referred to as “antisense technology”.

The term “hybridization,” as used herein, means hydrogen bonding, whichcan be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases. For example,adenine and thymine, and guanine and cytosine, respectively, arecomplementary nucleobases (often referred to in the art simply as“bases”) that pair through the formation of hydrogen bonds.“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleotides. For example, if a nucleotide at acertain position of an oligonucleotide is capable of hydrogen bondingwith a nucleotide in a target nucleic acid molecule, then theoligonucleotide and the target nucleic acid are considered to becomplementary to each other at that position. The oligonucleotide andthe target nucleic acid are complementary to each other when asufficient number of corresponding positions in each molecule areoccupied by nucleotides that can hydrogen bond with each other. Thus,“specifically hybridizable” is used to indicate a sufficient degree ofcomplementarity or precise pairing such that stable and specific bindingoccurs between the oligonucleotide and the target nucleic acid.

It is understood in the art that the sequence of an antisenseoligonucleotide need not be 100% complementary to that of its targetnucleic acid to be specifically hybridizable. An antisenseoligonucleotide is specifically hybridizable when (a) binding of theoligonucleotide to the target nucleic acid interferes with the normalfunction of the target nucleic acid, and (b) there is sufficientcomplementarity to avoid non-specific binding of the antisenseoligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under conditions in which in vitroassays are performed or under physiological conditions for in vivoassays or therapeutic uses.

Stringency conditions in vitro are dependent on temperature, time, andsalt concentration (see, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989)).Typically, conditions of high to moderate stringency are used forspecific hybridization in vitro, such that hybridization occurs betweensubstantially similar nucleic acids, but not between dissimilar nucleicacids. Specific hybridization conditions are hybridization in 5×SSC(0.75 M sodium chloride/0.075 M sodium citrate) for 1 hour at 40° C.with shaking, followed by washing 10 times in 1×SSC at 40° C. and 5times in 1×SSC at room temperature. Oligonucleotides that specificallyhybridize to a target nucleic acid can be identified by recovering theoligonucleotides from the oligonucleotide/target hybridization duplexes(e.g., by boiling) and sequencing the recovered oligonucleotides.

In vivo hybridization conditions consist of intracellular conditions(e.g., physiological pH and intracellular ionic conditions) that governthe hybridization of antisense oligonucleotides with target sequences.In vivo conditions can be mimicked in vitro by relatively low stringencyconditions, such as those used in the RiboTAG™ technology describedbelow. For example, hybridization can be carried out in vitro in 2×SSC(0.3 M sodium chloride/0.03 M sodium citrate), 0.1% SDS at 37° C. A washsolution containing 4×SSC, 0.1% SDS can be used at 37° C., with a finalwash in 1×SSC at 45° C.

The specific hybridization of an antisense molecule with its targetnucleic acid can interfere with the normal function of the targetnucleic acid. For a target DNA nucleic acid, antisense technology candisrupt replication and transcription. For a target RNA nucleic acid,antisense technology can disrupt, for example, translocation of the RNAto the site of protein translation, translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and catalyticactivity of the RNA. The overall effect of such interference with targetnucleic acid function is, in the case of a nucleic acid encoding P2X₃,modulation of the expression of P2X₃. In the context of the presentinvention, “modulation” means a decrease in the expression of a gene(e.g., due to inhibition of transcription) and/or a decrease in cellularlevels of the protein (e.g., due to inhibition of translation).

Identification of Target Sequences for P2X₃ Antisense Oligonucleotides

Antisense oligonucleotides are preferably directed at specific targetswithin a nucleic acid molecule. The process of “targeting” an antisenseoligonucleotide to a particular nucleic acid usually begins with theidentification of a nucleic acid sequence whose function is to bemodulated. This nucleic acid sequence can be, for example, a gene (ormRNA transcribed from the gene) whose expression is associated with aparticular disorder or disease state.

The targeting process also includes the identification of a site orsites within the target nucleic acid molecule where an antisenseinteraction can occur such that the desired effect, e.g., detection ofP2X₃ mRNA or modulation of P2X₃ expression, will result. Traditionally,preferred target sites for antisense oligonucleotides have included theregions encompassing the translation initiation or termination codon ofthe open reading frame (ORF) of the gene. In addition, the ORF has beentargeted effectively in antisense technology, as have the 5′ and 3′untranslated regions. Furthermore, antisense oligonucleotides have beensuccessfully directed at intron regions and intron-exon junctionregions.

Simple knowledge of the sequence and domain structure (e.g., thelocation of translation initiation codons, exons, or introns) of atarget nucleic acid, however, is generally not sufficient to ensure thatan antisense oligonucleotide directed to a specific region willeffectively bind to and modulate the function of the target nucleicacid. In its native state, an mRNA molecule is folded into complexsecondary and tertiary structures, and sequences that are on theinterior of such structures are inaccessible to antisenseoligonucleotides. For maximal effectiveness, antisense oligonucleotidescan be directed to regions of a target mRNA that are most accessible,i.e., regions at or near the surface of a folded mRNA molecule.

Accessible regions of an mRNA molecule can be identified by methodsknown in the art, including the use of RiboTAG™ technology. Thistechnology is disclosed in PCT application number SE01/02054. In theRiboTAG™ method, also known as mRNA Accessible Site Tagging (MAST),oligonucleotides that can interact with a test mRNA in its native state(i.e., under physiological conditions) are selected and sequenced, thusleading to the identification of regions within the test mRNA that areaccessible to antisense molecules. In a version of the RiboTAG™protocol, the test mRNA is produced by in vitro transcription and isthen immobilized, for example by covalent or non-covalent attachment toa bead or a surface (e.g., a magnetic bead). The immobilized test mRNAis then contacted by a population of oligonucleotides, wherein a portionof each oligonucleotide contains a different, random sequence.Oligonucleotides that can hybridize to the test mRNA under conditions oflow stringency are separated from the remainder of the population (e.g.,by precipitation of the magnetic beads). The selected oligonucleotidesthen can be amplified and sequenced; these steps of the protocol arefacilitated if the random sequences within each oligonucleotide areflanked on one or both sides by known sequences that can serve as primerbinding sites for PCR amplification.

In general, oligonucleotides that are useful in RiboTAG™ technologycontain between 15 and 18 random bases, flanked on either side by knownsequences. These oligonucleotides are contacted by the test mRNA underconditions that do not disrupt the native structure of the mRNA (e:g.,in the presence of medium pH buffering, salts that modulate annealing,and detergents and/or carrier molecules that minimize non-specificinteractions). Typically, hybridization is carried out at 37 to 40° C.,in a solution containing 1× to 5×SSC and about 0.1% SDS. Non-specificinteractions can be minimized further by blocking the known sequence(s)in each oligonucleotide with the primers that will be used for PCRamplification of the selected oligonucleotides.

As described herein, accessible regions of the nucleic acids encodinghuman and rat P2X₃ have been mapped. Thus, antisense oligonucleotides ofthe invention can specifically hybridize within one or more accessibleregions defined by: nucleotides 68 through 88, 209 through 230, 235through 247, 285 through 296, 346 through 355, 383 through 406, 490through 512, 530 through 543, 553 through 565, 649 through 658, 665through 679, 727 through 739, 756 through 779, 817 through 856, 874through 912, 959 through 991, 1028 through 1050, 1087 through 1116, 1145through 1177, 1237 through 1256, 1266 through 1281, 1297 through 1307,1314 through 1334, 1339 through 1359, 1434 through 1463, 1523 through1535, 1630 through 1646, 1677 through 1688, or 1729 through 1741 of SEQID NO:1. Particularly useful antisense oligonucleotides include thosethat specifically hybridize within accessible regions defined bynucleotides 383 through 406 (e.g., 384 through 397), 756 through 779(e.g., 766 through 775), 490 through 512 (e.g., 495 through 510), 727through 739 (e.g., 732 through 736), 1434 through 1463, 1237 through1256, 959 through 991, 1028 through 1050, 817 through 856, 553 through565, 285 through 296, 209 through 230, or 1145 through 1177 of SEQ IDNO:1.

Antisense oligonucleotides also can specifically hybridize withinaccessible regions defined by: nucleotides 7 through 29, 95 through 105,207 through 217, 221 through 240, 248 through 258, 278 through 293, 338through 365, 471 through 482, 486 through 502, 544 through 562, 747through 761, 784 through 796, 815 through 850, 865 through 879, 883through 905, 922 through 932, 953 through 968, 985 through 1000, 1033through 1044, 1156 through 1170, 1239 through 1261, 1297 through 1314,or 1411 through 1439 of SEQ ID NO:2. Particularly useful antisenseoligonucleotides include those that specifically hybridize withinaccessible regions defined by nucleotides 953 through 968 (e.g., 957through 967), 1297 through 1315 (e.g., 1297 through 1301), 815 through850 (e.g., 817 through 823), 747 through 761, 985 through 1000, 486through 502, 338 through 365, 278 through 293, 544 through 562, or 221through 240 of SEQ ID NO:2.

Non-limiting examples of such antisense oligonucleotides include thefollowing nucleotide sequences: 5′-GAC ACG TCC ATG ACT CTG TTG G-3′ (SEQID NO:3); 5′-GAG GTT TCC CTT CTC AAA-3′ (SEQ ID NO:4); 5′-TGT CCT TGTCGG TGA GGT TAG-3′ (SEQ ID NO:5); 5′-GTA GAC TGC TTC TCC ACA GTG-3′ (SEQID NO:6); 5′-CTC CTC ACT CTC TGG GCA-3′ (SEQ ID NO:7); and 5′-GTC CCTGGC TGT CAG GTT GGG A-3′ (SEQ ID NO:8).

It should be noted that an antisense oligonucleotide may consistessentially of a nucleotide sequence that specifically hybridizes withan accessible region set out above. Such antisense oligonucleotides,however, may contain additional flanking sequences of 5 to 10nucleotides at either end. Flanking sequences can include, for example,additional sequence of the target nucleic acid or primer sequence.

For maximal effectiveness, further criteria can be applied to the designof antisense oligonucleotides. Such criteria are well known in the art,and are widely used, for example, in the design of oligonucleotideprimers. These criteria include the lack of predicted secondarystructure of a potential antisense oligonucleotide, an appropriate G andC nucleotide content (e.g., approximately 50%), and the absence ofsequence motifs such as single nucleotide repeats (e.g., GGGG runs).

P2X₃ Antisense Oligonucleotides

Once one or more target sites have been identified, antisenseoligonucleotides can be synthesized that are sufficiently complementaryto the target (i.e., that hybridize with sufficient strength andspecificity to give the desired effect). In the context of the presentinvention, the desired effect is the modulation of P2X₃ expression suchthat cellular P2X₃ levels are reduced. The effectiveness of an antisenseoligonucleotide to modulate expression of a target nucleic acid can beevaluated by measuring levels of the mRNA or protein products of thetarget nucleic acid (e.g., by Northern blotting, RT-PCR, Westernblotting, ELISA, or immunohistochemical staining).

In some embodiments, it may be useful to target multiple accessibleregions of a target nucleic acid. In such embodiments, multipleantisense oligonucleotides can be used that each specifically hybridizeto a different accessible region. Multiple antisense oligonucleotidescan be used together or sequentially.

The antisense oligonucleotides in accordance with this invention can befrom about 10 to about 50 nucleotidesin length (e.g., 12 to 40, 14 to30, or 15 to 25 nucleotides in length). Antisense oligonucleotides thatare 15 to 23 nucleotides in length are particularly useful. However, anantisense oligonucleotide containing even fewer than 10 nucleotides (forexample, a portion of one of the preferred antisense oligonucleotides)is understood to be included within the present invention so long as itdemonstrates the desired activity of inhibiting expression of the P2X ₃purinoreceptor.

An “antisense oligonucleotide” can be an oligonucleotide as describedherein. The term “oligonucleotide” refers to an oligomer or polymer ofribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or analogsthereof. This term includes oligonucleotides composed of naturallyoccurring nucleobases, sugars and covalent internucleoside (backbone)linkages, as well as oligonucleotides having non-naturally occurringportions which function similarly. Such modified or substitutedoligonucleotides are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for a nucleic acid target, and increased stability inthe presence of nucleases.

While antisense oligonucleotides are a preferred form of antisensecompounds, the present invention includes other oligomeric antisensecompounds, including but not limited to, oligonucleotide analogs such asthose described below. As is known in the art, a nucleoside is abase-sugar combination, wherein the base portion is normally aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. Nucleotides are nucleosidesthat further include a phosphate group covalently linked to the sugarportion of the nucleoside. For those nucleosides that include apentofuranosyl sugar, the phosphate group can be linked to either the2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides,the phosphate groups covalently link adjacent nucleosides to one anotherto form a linear polymeric compound. The respective ends of this linearpolymeric structure can be further joined to form a circular structure,although linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

P2X₃ antisense oligonucleotides that are useful in the present inventioninclude oligonucleotides containing modified backbones or non-naturalinternucleoside linkages. As defined herein, oligonucleotides havingmodified backbones include those that have a phosphorus atom in thebackbone and those that do not have a phosphorus atom in the backbone.For the purposes of this specification, and as sometimes referenced inthe art, modified oligonucleotides that do not have a phosphorus atom intheir internucleoside backbone also can be considered to beoligonucleotides.

Modified oligonucleotide backbones can include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkylphosphonates (e.g., 3′-alkylene phosphonates and chiral phosphonates),phosphinates, phosphoramidates (e.g., 3′-amino phosphoramidate andaminoalkylphosphoramidates), thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, as well as 2′-5′ linkedanalogs of these, and those having inverted polarity wherein theadjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to5′-2′. Various salts, mixed salts and free acid forms are also included.References that teach the preparation of such modified backboneoligonucleotides are provided, for example, in U.S. Pat. Nos. 4,469,863and 5,750,666.

P2X₃ antisense molecules with modified oligonucleotide backbones that donot include a phosphorus atom therein can have backbones that are formedby short chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These include those having morpholino linkages (formed in part from thesugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxideand sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts. References thatteach the preparation of such modified backbone oligonucleotides areprovided, for example, in U.S. Pat. Nos. 5,235,033 and 5,596,086.

In another embodiment, a P2X₃ antisense compound can be anoligonucleotide analog, in which both the sugar and the internucleosidelinkage (i.e., the backbone) of the nucleotide units are replaced withnovel groups, while the base units are maintained for hybridization withan appropriate nucleic acid target. One such oligonucleotide analog thathas been shown to have excellent hybridization properties is referred toas a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone ofan oligonucleotide is replaced with an amide containing backbone (e.g.,an aminoethylglycine backbone). The nucleobases are retained and arebound directly or indirectly to aza nitrogen atoms of the amide portionof the backbone. References that teach the preparation of such modifiedbackbone oligonucleotides are provided, for example, in Nielsen et al.,Science 254:1497-1500 (1991), and in U.S. Pat. No. 5,539,082.

Other useful P2X₃ antisense oligonucleotides can have phosphorothioatebackbones and oligonucleosides with heteroatom backbones, and inparticular CH₂NHOCH₂, CH₂N(CH₃)OCH₂, CH₂ON(CH₃)CH₂, CH₂N(CH₃)N(CH₃)CH₂,and ON(CH₃)CH₂CH₂ (wherein the native phosphodiester backbone isrepresented as OPOCH₂) as taught in U.S. Pat. No.5,489,677, and theamide backbones disclosed in U.S. Pat. No. 5,602,240.

Substituted sugar moieties also can be included in modifiedoligonucleotides. P2X₃ antisense oligonucleotides of the invention cancomprise one or more of the following at the 2′ position: OH; F; O—, S—,or N-alkyl; O—, S—, or N-alkenyl; O—, S—, or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Useful modifications also can include O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(C₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. Inaddition, oligonucleotides can comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, groups for improving thepharmacokinetic or pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Other useful modificationsinclude an alkoxyalkoxy group, e.g., 2′-methoxyethoxy (2′-OCH₂CH₂OCH₃),a dimethylaminooxyethoxy group (2′-O(CH₂)₂ON(CH₃)₂), or adimethylamino-ethoxyethoxy group (2′-OCH₂OCH₂N(CH₂)₂). Othermodifications can include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), or 2′-fluoro (2′-F). Similar modifications also canbe made at other positions within the oligonucleotide, such as the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides, and the 5′ position of the 5′ terminal nucleotide.Oligonucleotides also can have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl group. References that teach thepreparation of such substituted sugar moieties include U.S. Pat. Nos.4,981,957 and 5,359,044.

Useful P2X₃ antisense oligonucleotides also can include nucleobasemodifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include the purine bases adenine (A) and guanine(G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).Modified nucleobases can include other synthetic and natural nucleobasessuch as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanineand 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanineand 3-deazaadenine. Other useful nucleobases include those disclosed,for example, in U.S. Pat. No. 3,687,808.

Certain nucleobase substitutions can be particularly useful forincreasing the binding affinity of the antisense oligonucleotides of theinvention. For example, 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6 to 1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, pp. 276-278, CRC Press, Boca Raton, Fla. (1993)). Otheruseful nucleobase substitutions include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines such as2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

Antisense oligonucleotides of the invention also can be modified bychemical linkage to one or more moieties or conjugates that enhance theactivity, cellular distribution or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties (e.g., a cholesterol moiety); cholic acid; a thioether moiety(e.g., hexyl-S-tritylthiol); a thiocholesterol moiety; an aliphaticchain (e.g., dodecandiol or undecyl residues); a phospholipid moiety(e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate); a polyamine or apolyethylene glycol chain; adamantane acetic acid; a palmityl moiety; oran octadecyl amine or hexylamino-carbonyl-oxycholesterol moiety. Thepreparation of such oligonucleotide conjugates is disclosed in, forexample, U.S. Pat. Nos. 5,218,105 and 5,214,136.

It is not necessary for all nucleobase positions in a given antisenseoligonucleotide to be uniformly modified. More than one of theaforementioned modifications can be incorporated into a singleoligonucleotide or even at a single nucleoside within anoligonucleotide. The present invention also includes antisenseoligonucleotides that are chimeric oligonucleotides. “Chimeric”antisense oligonucleotides can contain two or more chemically distinctregions, each made up of at least one monomer unit (e.g., a nucleotidein the case of an oligonucleotide). Chimeric oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer, for example, increased resistance to nuclease degradation,increased cellular uptake, and/or increased affinity for the targetnucleic acid. For example, a region of a chimeric oligonucleotide canserve as a substrate for an enzyme such as RNase H, which is capable ofcleaving the RNA strand of an RNA:DNA duplex such as that formed betweena target mRNA and an antisense oligonucleotide. Cleavage of such aduplex by RNase H, therefore, can greatly enhance the effectiveness ofan antisense oligonucleotide.

The P2X₃ antisense oligonucleotides of the invention are synthesized invitro and do not include antisense compositions of biological origin,except for oligonucleotides that comprise the subject antisenseoligonucleotides and that have been purified from or isolated frombiological material. Antisense oligonucleotides used in accordance withthis invention can be conveniently produced through the well-knowntechnique of solid phase synthesis. Equipment for such synthesis iscommercially available from several vendors including, for example,Applied Biosystems (Foster City, Calif.). Any other means for suchsynthesis known in the art additionally or alternatively can beemployed. Similar techniques also can be used to prepare modifiedoligonucleotides such as phosphorothioates or alkylated derivatives.

Antisense Preparations and Methods for Use

The antisense oligonucleotides of the invention are useful for researchand diagnostics, and for therapeutic use. For example, assays based onhybridization of antisense oligonucleotides to nucleic acids encodingP2X₃ can be used to evaluate levels of P2X₃ in a tissue sample.Hybridization of the antisense oligonucleotides of the invention with anucleic acid encoding P2X₃ can be detected by means known in the art.Such means can include conjugation of an enzyme to the antisenseoligonucleotide, radiolabeling of the antisense oligonucleotide, or anyother suitable means of detection.

Those of skill in the art can harness the specificity and sensitivity ofantisense technology for therapeutic use. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals, including humans. For therapeutic methods, the cellsor tissues are typically within a vertebrate (e.g., a mammal such as ahuman.

The invention provides therapeutic methods for treating conditionsarising from abnormal expression (e.g., over-production) of the P2X₃purinoreceptor. By these methods, antisense oligonucleotides inaccordance with the invention are administered to a subject (e.g., ahuman) suspected of having a disease or disorder (e.g., chronic pain,overactive bladder, or irritable bowel syndrome) that can be alleviatedby modulating the expression of P2X₃. Typically, one or more antisenseoligonucleotides can be administered to a subject suspected of having adisease or condition associated with the expression of P2X₃. Theantisense oligonucleotide can be in a pharmaceutically acceptablecarrier or diluent, and can be administered in amounts and for periodsof time that will vary depending upon the nature of the particulardisease, its severity, and the subject's overall condition. Typically,the antisense oligonucleotide is administered in an inhibitory amount(i.e., in an amount that is effective for inhibiting the production ofP2X₃ in the cells or tissues contacted by the antisenseoligonucleotides). The antisense oligonucleotides and methods of theinvention also can be used prophylactically, e.g., to minimize pain in asubject known to have high levels of P2X₃.

The ability of a P2X₃ antisense oligonucleotide to inhibit expressionand/or production of P2X₃ can be assessed, for example, by measuringlevels of P2X₃ mRNA or protein in a subject before and after treatment.Methods for measuring mRNA and protein levels in tissues or biologicalsamples are well known in the art. If the subject is a research animal,for example, P2X₃ levels in the brain can be assessed by in situhybridization or immunostaining following euthanasia. Indirect methodscan be used to evaluate the effectiveness of P2X₃ antisenseoligonucleotides in live subjects. For example, reduced expression ofP2X₃ can be inferred from reduced sensitivity to painful stimuli. Asdescribed in the Examples below, animal models can be used to study thedevelopment, maintenance, and relief of chronic neuropathic orinflammatory pain. Animals subjected to these models generally developthermal hyperalgesia (i.e., an increased response to a stimulus that isnormally painful) and/or allodynia (i.e., pain due to a stimulus that isnot normally painful). Sensitivity to mechanical and thermal stimuli canbe assessed (see Bennett, Methods in Pain Research, pp. 67-91, Kruger,Ed. (2001)) to evaluate the effectiveness of P2X₃ antisense treatment.

Methods for formulating and subsequently administering therapeuticcompositions are well known to those skilled in the art. See, forexample, Remington, The Science and Practice of Pharmacy, 20^(th) Ed.,Gennaro & Gennaro, eds., Lippincott, Williams & Wilkins (2000). Dosingis generally dependent on the severity and responsiveness of the diseasestate to be treated, with the course of treatment lasting from severaldays to several months, or until a cure is effected or a diminution ofthe disease state is achieved. Persons of ordinary skill in the artroutinely determine optimum dosages, dosing methodologies and repetitionrates. Optimum dosages can vary depending on the relative potency ofindividual oligonucleotides, and can generally be estimated based onEC₅₀ found to be effective in in vitro and in vivo animal models.Typically, dosage is from 0.01 μg to 100 g per kg of body weight, andmay be given once or more daily, weekly, or even less often. Followingsuccessful treatment, it may be desirable to have the patient undergomaintenance therapy to prevent the recurrence of the disease state.

The present invention provides pharmaceutical compositions andformulations that include the P2X₃ antisense oligonucleotides of theinvention. P2X₃ antisense oligonucleotides therefore can be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecular structures, or mixtures of oligonucleotides such as, forexample, liposomes, receptor targeted molecules, or oral, rectal,topical or other formulations, for assisting in uptake, distributionand/or absorption.

A “pharmaceutically acceptable carrier” (also referred to herein as an“excipient”) is a pharmaceutically acceptable solvent, suspending agent,or any other pharmacologically inert vehicle for delivering one or moretherapeutic compounds (e.g., P2X₃ antisense oligonucleotides) to asubject. Pharmaceutically acceptable carriers can be liquid or solid,and can be selected with the planned manner of administration in mind soas to provide for the desired bulk, consistency, and other pertinenttransport and chemical properties, when combined with one or more oftherapeutic compounds and any other components of a given pharmaceuticalcomposition. Typical pharmaceutically acceptable carriers that do notdeleteriously react with nucleic acids include, by way of example andnot limitation: water; saline solution; binding agents (e.g.,polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose and other sugars, gelatin, or calcium sulfate); lubricants(e.g., starch, polyethylene glycol, or sodium acetate); disintegrates(e.g., starch or sodium starch glycolate); and wetting agents (e.g.,sodium lauryl sulfate).

The pharmaceutical compositions of the present invention can beadministered by a number of methods depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be, for example, topical (e.g., transdermal,ophthalmic, or intranasal); pulmonary (e.g., by inhalation orinsufflation of powders or aerosols); oral; or parenteral (e.g., bysubcutaneous, intrathecal, intraventricular, intramuscular, orintraperitoneal injection, or by intravenous drip). Administration canbe rapid (e.g., by injection) or can occur over a period of time (e.g.,by slow infusion or administration of slow release formulations). Fortreating tissues in the central nervous system, antisenseoligonucleotides can be administered by injection or infusion into thecerebrospinal fluid, preferably with one or more agents capable ofpromoting penetration of the antisense oligonucleotide across theblood-brain barrier.

Formulations for topical administration of antisense oligonucleotidesinclude, for example, sterile and non-sterile aqueous solutions,non-aqueous solutions in common solvents such as alcohols, or solutionsin liquid or solid oil bases. Such solutions also can contain buffers,diluents and other suitable additives. Pharmaceutical compositions andformulations for topical administration can include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquids,and powders. Coated condoms, gloves and the like also may be useful.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions and formulations for oral administration include, forexample, powders or granules, suspensions or solutions in water ornon-aqueous media, capsules, sachets, or tablets. Such compositions alsocan incorporate thickeners, flavoring agents, diluents, emulsifiers,dispersing aids, or binders. Oligonucleotides with at least one2′-O-methoxyethyl modification (described above) may be particularlyuseful for oral administration.

Compositions and formulations for parenteral, intrathecal orintraventricular administration can include sterile aqueous solutions,which also can contain buffers, diluents and other suitable additives(e.g., penetration enhancers, carrier compounds and otherpharmaceutically acceptable carriers).

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, aqueous suspensions, andliposome-containing formulations. These compositions can be generatedfrom a variety of components that include, for example, preformedliquids, self-emulsifying solids and self-emulsifying semisolids.Emulsions are often biphasic systems comprising of two immiscible liquidphases intimately mixed and dispersed with each other; in general,emulsions are either of the water-in-oil (w/o) or oil-in-water (o/w)variety. Emulsion formulations have been widely used for oral deliveryof therapeutics due to their ease of formulation and efficacy ofsolubilization, absorption, and bioavailability.

Liposomes are vesicles that have a membrane formed from a lipophilicmaterial and an aqueous interior that can contain the antisensecomposition to be delivered. Liposomes can be particularly useful fromthe standpoint of drug delivery due to their specificity and theduration of action they offer. Liposome compositions can be formed, forexample, from phosphatidylcholine, dimyristoyl phosphatidylcholine,dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, ordioleoyl phosphatidylethanolamine. Numerous lipophilic agents arecommercially available, including Lipofectin® (Invitrogen/LifeTechnologies, Carlsbad, Calif.) and Effectene™ (Qiagen, Valencia,Calif.).

The P2X₃ antisense oligonucleotides of the invention further encompassany pharmaceutically acceptable salts, esters, or salts of such esters,or any other compound which, upon administration to an animal includinga human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof Accordingly, forexample, the invention provides pharmaceutically acceptable salts ofP2X₃ antisense oligonucleotides, prodrugs and pharmaceuticallyacceptable salts of such prodrugs, and other bioequivalents. The term“prodrug” indicates a therapeutic agent that is prepared in an inactiveform and is converted to an active form (i.e., drug) within the body orcells thereof by the action of endogenous enzymes or other chemicalsand/or conditions. The term “pharmaceutically acceptable salts” refersto physiologically and pharmaceutically acceptable salts of theoligonucleotides of the invention (i.e., salts that retain the desiredbiological activity of the parent oligonucleotide without impartingundesired toxicological effects). Examples of pharmaceuticallyacceptable salts of oligonucleotides include, but are not limited to,salts formed with cations (e.g., sodium, potassium, calcium, orpolyamines such as spermine); acid addition salts formed with inorganicacids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, or nitric acid); salts formed with organic acids (e.g.,acetic acid, citric acid, oxalic acid, palmitic acid, or fumaric acid);and salts formed from elemental anions (e.g., chlorine, bromine, andiodine).

Pharmaceutical compositions containing the antisense oligonucleotides ofthe present invention also can incorporate penetration enhancers thatpromote the efficient delivery of nucleic acids, particularlyoligonucleotides, to the skin of animals. Penetration enhancers canenhance the diffusion of both lipophilic and non-lipophilic drugs acrosscell membranes. Penetration enhancers can be classified as belonging toone of five broad categories, i.e., surfactants (e.g., sodium laurylsulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetylether); fatty acids (e.g., oleic acid, lauric acid, myristic acid,palmitic acid, and stearic acid); bile salts (e.g., cholic acid,dehydrocholic acid, and deoxycholic acid); chelating agents (e.g.,disodium ethylenediaminetetraacetate, citric acid, and salicylates); andnon-chelating non-surfactants (e.g., unsaturated cyclic ureas).

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense oligonucleotides and (b) one ormore other agents that function by a non-antisense mechanism. Forexample, anti-inflammatory drugs, including but not limited tononsteroidal anti-inflammatory drugs and corticosteroids, and antiviraldrugs, including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, can be included in compositions of the invention. Othernon-antisense agents (e.g., chemotherapeutic agents) are also within thescope of this invention. Such combined compounds can be used together orsequentially.

The antisense compositions of the present invention additionally cancontain other adjunct components conventionally found in pharmaceuticalcompositions. Thus, the compositions also can include compatible,pharmaceutically active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, oradditional materials useful in physically formulating various dosageforms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. Furthermore, the composition can be mixed withauxiliary agents, e.g., lubricants, preservatives, stabilizers, wettingagents, emulsifiers, salts for influencing osmotic pressure, buffers,colorings, flavorings, and aromatic substances. When added, however,such materials should not unduly interfere with the biologicalactivities of the antisense components within the compositions of thepresent invention. The formulations can be sterilized and, if desired,and the like which do not deleteriously interact with the nucleicacid(s) of the formulation.

The pharmaceutical formulations of the present invention, which can bepresented conveniently in unit dosage form, can be prepared according toconventional techniques well known in the pharmaceutical industry. Suchtechniques include the step of bringing into association the activeingredients (e.g., the P2X₃ antisense oligonucleotides of the invention)with the desired pharmaceutical carrier(s) or excipient(s). Typically,the formulations can be prepared by uniformly bringing the activeingredients into intimate association with liquid carriers or finelydivided solid carriers or both, and then, if necessary, shaping theproduct. Formulations can be sterilized if desired, provided that themethod of sterilization does not interfere with the effectiveness of theantisense oligonucleotide contained in the formulation.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention also can be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsfurther can contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol, and/or dextran. Suspensions also can contain stabilizers.

Nucleic Acid Constructs

Nucleic acid constructs (e.g., a plasmid vector) are capable oftransporting a nucleic acid into a host cell. Suitable host cellsinclude prokaryotic or eukaryotic cells (e.g., bacterial cells such asE. coli, insect cells, yeast cells, and mammalian cells). Someconstructs are capable of autonomously replicating in a host cell intowhich they are introduced (e.g., bacterial vectors having a bacterialorigin of replication and episomal mammalian vectors). Other vectors(e.g., non-episomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell and are replicated withthe host genome.

Nucleic acid constructs can be, for example, plasmid vectors or viralvectors (e.g., replication defective retroviruses, adenoviruses, andadeno-associated viruses). Nucleic acid constructs include one or moreregulatory sequences operably linked to the nucleic acid of interest(e.g., a nucleic acid encoding a transcript that specifically hybridizesto a P2X₃ mRNA in its native form). With respect to regulatory elements,“operably linked” means that the regulatory sequence and the nucleicacid of interest are positioned such that nucleotide sequence istranscribed (e.g., when the vector is introduced into the host cell).

Regulatory sequences include promoters, enhancers and other expressioncontrol elements (e.g., polyadenylation signals). (See, e.g., Goeddel,Gene Expression Technology: Methods in Enzymology, 185, Academic Press,San Diego, Calif. (1990)). Regulatory sequences include those thatdirect constitutive expression of a nucleotide sequence in many types ofhost cells and that direct expression of the nucleotide sequence only incertain host cells (e.g., cell type or tissue-specific regulatorysequences).

Articles of Manufacture

Antisense oligonucleotides of the invention can be combined withpackaging material and sold as kits of reducing P2X₃ expression.Components and methods for producing articles of manufacture are wellknown. The articles of manufacture may combine one or more of theantisense oligonucleotides set out in the above sections. In addition,the article of manufacture further may include buffers, hybridizationreagents, or other control reagents for reducing or monitoring reducedP2X₃ expression. Instructions describing how the antisenseoligonucleotides are effective for reducing P2X₃ expression can beincluded in such kits.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Materials and Methods

Determination of Accessible Sites within the P2X₃ mRNA and Design ofP2X₃ Antisense Oligonucleotides

Antisense oligonucleotides were designed to be targeted to accessibleregions of the rat or human P2X₃ mRNA (as determined by the RiboTAG™method). Accessible regions of rat and human P2X₃ are shown in Table 1.Antisense oligonucleotides were commercially synthesized (MidlandCertified Reagent Company, Midland, Tex.) and purified prior toinjection. TABLE 1 Accessible sequences within P2X₃ mRNA Rat P2X₃ HumanP2X₃ Start End Start End 68 88 7 29 209 230 95 105 235 247 207 217 285296 221 240 346 355 248 258 383 406 278 293 490 512 338 365 530 543 471482 553 565 486 502 649 658 544 562 665 679 747 761 727 739 784 796 756779 815 850 817 856 865 879 874 912 883 905 959 991 922 932 1028 1050953 968 1087 1116 985 1000 1145 1177 1033 1044 1237 1256 1156 1170 12661281 1239 1261 1297 1307 1297 1314 1314 1334 1411 1439 1339 1359 14341463 1523 1535 1630 1646 1677 1688 1729 1741Methods for Evaluating Pain in Rats Treated with Antisense P2X₃

Two different models of chronic pain were used to evaluate the effectsof P2X₃ knock-down by intrathecally administered antisenseoligonucleotides. Both models included the following six steps(described in greater detail below):

(1) spinal catheterization;

(2) nociceptive testing (baseline);

(3) induction of chronic neuropathic or inflammatory pain;

(4) nociceptive testing (post-injury);

(5) antisense injection; and

(6) nociceptive testing (post-treatment).

Spinal Catheterization: Male Sprague Dawley rats weighing between 120and 150 g were obtained from Harlan (Indianapolis, Ind.). Rats weredeeply anesthetized with a mixture containing 75 mg/kg ketamine, 5 mg/kgxylazine, and 1 mg/kg acepromazine, and a catheter (8.5 cm; PE-10) waspassed to the lumbosacral intrathecal space through an incision in thedura over the atlantooccipital joint. Animals were allowed to recoverfor 3 days before being subjected to models of chronic pain.

Mechanical Nociceptive Testing: Baseline, post-injury, andpost-treatment values for mechanical sensitivity were evaluated withcalibrated monofilaments (von Frey filaments) according to the up-downmethod (Chaplan, et al. (1994) J. Neurosci. Methods 53:55-63). Animalswere placed on a wire mesh platform and allowed to acclimate to theirsurroundings for a minimum of 10 minutes before testing. Filaments ofincreasing force were sequentially applied to the plantar surface of thepaw just to the point of bending, and held for three seconds. Thebehavioral endpoint of the stimulus (achieved when the stimulus was ofsufficient force) was the point at which the animal would lick, withdrawand/or shake the paw. The force or pressure required to cause a pawwithdrawal was recorded as a measure of threshold to noxious mechanicalstimuli for each hind-paw. The mean and standard error of the mean (SEM)were determined for each hind-paw in each animal in each treatmentgroup. Repeated measures ANOVA followed by the Bonferonni post-hoc testwere used to determine significance. Since this stimulus is normally notconsidered painful and rats do not normally respond to filaments in therange selected, significant injury-induced increases in responsivenessin this test were interpreted as a measure of mechanical allodynia.

Thermal Nociceptive Testing: Baseline, post-injury, and post-treatmentthermal sensitivities were determined by measuring withdrawal latenciesin response to radiant heat stimuli delivered to the plantar surface ofthe hind-paws (Hargreaves et al. (1988), Pain 32:77-88). Animals wereplaced on a plexiglass platform and allowed to acclimate for a minimumof 10 minutes. A radiant heat source was directed to the plantarsurface, and the time to withdrawal was measured. For each paw, thewithdrawal latency was determined by averaging three measurementsseparated by at least 5 minutes. The heating device was set toautomatically shut off after a programmed period of time to avoid damageto the skin of unresponsive animals. The data were analyzed usingrepeated measures ANOVA followed by the Bonferonni post-hoc test.Significant injury-induced increases in thermal response latencies wereconsidered to be a measure of thermal hyperalgesia since the stimulus isnormally in the noxious range.

Induction of Chronic Neuropathic Pain: The Spinal Nerve Ligation (SNL)model (Kim and Chung (1992) Pain 50:355-363) was used to induce chronicneuropathic pain. Rats were anesthetized with isoflurane, the L5transverse process was removed, and the L5 and L6 spinal nerves weretightly ligated with 6-0 silk suture. The wound was then closed withinternal sutures and external staples. Following surgery, animals werekept on a warming blanket and were periodically turned and carefullyobserved until complete recovery from anesthesia was obtained. Shamsurgery consisted of removing the transverse process and exposing the L5spinal nerve without ligating. All operations were performed on the leftside.

Induction of Chronic Inflammation: The complete freunds adjuvant (CFA)model of chronic peripheral inflammation was utilized (see, for example,Hylden et al. (1989) Pain 37:229-243). Rats under isoflurane anesthesiareceived an injection of CFA (75 μl) into the left hindpaw using asterile 1.0 ml syringe. A separate population of control rats wassubjected to unilateral injection of saline.

Antisense Injection: Oligonucleotides were dissolved in dH₂O anddelivered into the intrathecal space in a volume of 5 μl per injectionas previously described (see, for example, Bilsky et al. (1996)Neurosci. Lett., 220:155-158; Bilsky et al. (1996) J. Pharmacol. Exp.Ther., 277:491-501; and Vanderah et al. (1994) Neuroreport.,5:2601-2605). Antisense oligonucleotides were administered twice dailyfor 3 to 4 days, beginning on the afternoon following post-injury(baseline) nociceptive testing. Antisense oligonucleotides included thesequences 5′-GAG GTT TCC CTT CTC AAA-3′, 5′-ATG TCC TTG TCG GTG AGG TTAGG-3′, and 5′-CTA GTC TTT GGG GTG AAC-3′, which specifically hybridizeto nucleotides 721 through 744, 747 through 770, and 1478 through 1495,respectively, of SEQ ID NO:1. Random oligonucleotides were used ascontrols. Random oligonucleotides were used as controls.

Immunolocalization of P2X₃

Spinal cord tissue was obtained post-mortem and immersion-fixedovernight in 4% paraformaldehyde. After fixation, the tissue was washedin phosphate buffered saline (PBS) for 2 to 3 days and stored in 10%sucrose solution. The spinal cord was sliced into 14 μm sections using acryostat. Slide-mounted tissue sections were incubated in blockingbuffer for 1 hour at room temperature, followed by incubation withprimary antisera (guinea pig anti-P2X₃, 1:5000) overnight at 4° C.Staining was visualized using biotinylated tiramine amplification aspreviously described (Vulchanova et al. (1997) Neuropharmacology36:1229-1242). For absorption control, the primary antisera wereincubated with the corresponding peptide antigen (10 μg/ml) prior toapplication to tissue sections.

Dorsal root ganglia were obtained post-mortem and frozen in liquidnitrogen. The frozen tissue was cut into 10 μm sections, which werethaw-mounted on cooled gelatin-coated slides. Sections were fixed withparaformaldehyde-picric acid fixative for 30 minutes immediately beforeprocessing for immunohistochemistry. The slide-mounted tissue sectionswere incubated in blocking buffer for 1 hour at room temperature,followed by incubation with primary antisera (guinea pig anti-P2X₃,1:500) overnight at 4° C. Slides were washed three times in PBS,incubated with secondary antisera for 1 hour at room temperature, washedagain and coverslipped. For absorption control, the primary antiserawere incubated with the corresponding peptide antigen (10 μg/ml) priorto applying to tissue sections. Staining was visualized with cyanine3.18-conjugated secondary antisera (Jackson ImmunoResearch, West Grove,Calif.).

Example 2 Antisense Knockdown of P2X3 in Rat Spinal Cord Supports a Rolein Chronic Neuropathic and Inflammatory Pain

Antisense oligonucleotides were designed by the RiboTAG™ method and usedto evaluate the role of P2X₃ in chronic pain. Thermal (radiant heat) andmechanical (von Frey) pain thresholds were obtained before and afterinduction of chronic pain (neuropathic or inflammatory, as described inExample 1, above). Antisense oligonucleotides or randomized controlswere delivered twice daily for 3 to 4 days, and thermal and mechanicalthresholds were reassessed.

FIG. 3A shows that animals were significantly more sensitive to thermalstimuli following nerve injury by spinal nerve ligation (as evidenced bythe decreases in their response thresholds compared to pre-injurybaseline (BL) and uninjured controls). Treatment with P2X₃ antisenseoligonucleotides between days 0 and 3 significantly relieved thesensitivity to thermal stimulation, as compared to treatment with acontrol vehicle. Cessation of treatment between days 3 and 15 caused areturn to maximal sensitivity, which was again relieved by theresumption of antisense treatment between days 15 and 18. Repeatinjections of vehicle only had no effect.

Similarly, FIG. 3B shows that nerve-injured animals also weresignificantly more sensitive to mechanical stimuli, and that P2X₃antisense treatment reversibly relieved this sensitivity.

FIG. 4A and FIG. 4B depict the results of analagous experiments using aseparate group of animals. As in the previous experiments, treatment ofchronic pain with P2X₃ antisense oligonucleotides reversibly alleviatedthe sensitivity to thermal and mechanical stimuli.

As shown in FIG. 5A and FIG. 5B, animals subjected to inflammation alsowere significantly more sensitive to thermal and mechanical stimuli (asevidenced by the decreases in their response thresholds compared topre-inflammation baseline (BL) and uninflamed controls).

Animals from several experiments were euthanized so that levels of P2X₃could be assessed by immunostaining. Rats treated with the P2X₃antisense oligonucleotides displayed lower levels of P2X₃ in dorsal hornspinal cord tissue than control animals treated with randomizedoligonucleotides.

Example 3 Immunolocalization of human P2X₃

P2X₃ immunoreactivity in human spinal cord was restricted to a bandcorresponding to inner lamina II (FIG. 6A and 6A′). In dorsal rootganglia, P2X₃ immunoreactivity was present in a subset of small neurons(FIG. 6B and 6B′). Specificity of the labeling in spinal cord and dorsalroot ganglia was demonstrated by absorption controls with the peptideantigen (A′ and B′, respectively). This localization of human P2X₃ wasidentical to the localization of rat P2X₃, which supports the pursuit ofP2X₃ as a therapeutic target for chronic pain.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An isolated antisense oligonucleotide consisting essentially of 10 to50 nucleotides, wherein said oligonucleotide specifically hybridizeswithin an accessible region, said region defined by nucleotides 68through 88, 209 through 230, 235 through 247, 285 through 296, 346through 355, 383 through 406, 490 through 512, 530 through 543, 553through 565, 649 through 658, 665 through 679, 727 through 739, 756through 779, 817 through 856, 874 through 912, 959 through 991, 1028through 1050, 1087 through 1116, 1145 through 1177, 1237 through 1256,1266 through 1281, 1297 through 1307, 1314 through 1334, 1339 through1359, 1434 through 1463, 1523 through 1535, 1630 through 1646, 1677through 1688, or 1729 through 1741 of SEQ ID NO:1, and wherein saidoligonucleotide inhibits the production of P2X₃.
 2. The isolatedantisense oligonucleotide of claim 1, wherein said oligonucleotidespecifically hybridizes within an accessible region defined bynucleotides 383 through 406, 756 through 779, 490 through 512, or 727through 739 of SEQ ID NO:1.
 3. The isolated antisense oligonucleotide ofclaim 2, wherein said oligonucleotide specifically hybridizes within anaccessible region defined by nucleotides 384 through 397, 766 through775, 495 through 510, or 732 through 736 of SEQ ID NO:1.
 4. The isolatedantisense oligonucleotide of claim 1, wherein said oligonucleotidespecifically hybridizes within an accessible region defined bynucleotides 1434 through 1463, 1237 through 1256, 959 through 991, or1028 through 1050 of SEQ ID NO:1.
 5. The isolated antisenseoligonucleotide of claim 1, wherein said oligonucleotide specificallyhybridizes within an accessible region defined by nucleotides 817through 856, 553 through 565, 285 through 296, 209 through 230, or 1145through 1177 of SEQ ID NO:1.
 6. The isolated antisense oligonucleotideof claim 1, wherein said oligonucleotide specifically hybridizes withinan accessible region defined by nucleotides 383 through 404, 721 through744, 747 through 770, or 1314 through 1344 of SEQ ID NO:1.
 7. Acomposition comprising the isolated antisense oligonucleotide ofclaim
 1. 8. The composition of claim 7, wherein said compositioncomprises a plurality of isolated antisense oligonucleotides, whereineach antisense oligonucleotide specifically hybridizes within adifferent accessible region.
 9. An isolated antisense oligonucleotideconsisting essentially of 10 to 50 nucleotides, wherein saidoligonucleotide specifically hybridizes within an accessible region,said region defined by nucleotides 7 through 29, 95 through 105, 207through 217, 221 through 240, 248 through 258, 278 through 293, 338through 365, 471 through 482, 486 through 502, 544 through 562, 747through 761, 784 through 796, 815 through 850, 865 through 879, 883through 905, 922 through 932, 953 through 968, 985 through 1000, 1033through 1044, 1156 through 1170, 1239 through 1261, 1297 through 1314,or 1411 through 1439 of SEQ ID NO:2, and wherein said isolated antisenseoligonucleotide inhibits the production of P2X₃.
 10. The isolatedantisense oligonucleotide of claim 9, wherein said antisenseoligonucleotide specifically hybridizes within an accessible regiondefined by nucleotides 953 through 968, 1297 through 1314, or 815through 850 of SEQ ID NO:2.
 11. The isolated antisense oligonucleotideof claim 10, wherein said antisense oligonucleotide specificallyhybridizes within an accessible region defined by nucleotides 957through 967, 1297 through 1301, or 817 through 823 of SEQ ID NO:2. 12.The isolated antisense oligonucleotide of claim 9, wherein saidantisense oligonucleotide specifically hybridizes within an accessibleregion defined by nucleotides 747 through 761, 985 through 1000, or 486through 502 of SEQ ID NO:2.
 13. The isolated antisense oligonucleotideof claim 9, wherein said antisense oligonucleotide specificallyhybridizes within an accessible region defined by nucleotides 338through 365, 278 through 293, 544 through 562, or 221 through 240 of SEQID NO:2.
 14. The isolated antisense oligonucleotide of claim 9, whereinsaid antisense oligonucleotide specifically hybridizes within anaccessible region defined by nucleotides 484 through 501 or 742 through762 of SEQ ID NO:2.
 15. A composition comprising the isolated antisenseoligonucleotide of claim
 9. 16. The composition of claim 15, whereinsaid composition comprises a plurality of isolated antisenseoligonucleotides, wherein each antisense oligonucleotide specificallyhybridizes with a different accessible region.
 17. An isolatedoligonucleotide consisting essentially of the sequence of SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. 18.A method of decreasing production of P2X₃ in cells or tissues,comprising contacting said cells or tissues with an antisenseoligonucleotide that specifically hybridizes within an accessible regionof P2X₃ provided that said accessible region is not a region defined bynucleotides 1279 through 1296 or 1315 through 1334 of SEQ ID NO:2. 19.The method of claim 18, wherein said contacting step results in aninhibition of pain sensory neurons.
 20. The method of claim 18, whereinsaid contacting step results in increased bladder capacity.
 21. Anucleic acid construct comprising a regulatory element operably linkedto a nucleic acid encoding a transcript, wherein said transcriptspecifically hybridizes within one or more accessible regions of P2X₃mRNA in its native form.
 22. A host cell comprising the nucleic acidconstruct of claim
 21. 23. An isolated antisense oligonucleotide thatspecifically hybridizes within an accessible region of P2X₃ mRNA in itsnative form, provided that said accessible region is not a regiondefined by nucleotides 1279 through 1296 or 1315 through 1334 of SEQ IDNO:2, and wherein said antisense oligonucleotide inhibits production ofP2X₃.